Department of Pharmacy, University of Naples Federico II, Naples, Italy; Endocannabinoid Research Group.

Abstract

BACKGROUND AND PURPOSE:

The non-psychotropic cannabinoid cannabichromene is known to activate the transient receptor potential ankyrin-type1 (TRPA1) and to inhibit endocannabinoid inactivation, both of which are involved in inflammatory processes. We examined here the effects of this phytocannabinoid on peritoneal macrophages and its efficacy in an experimental model of colitis.

EXPERIMENTAL APPROACH:

Murine peritoneal macrophages were activated in vitro by LPS. Nitrite levels were measured using a fluorescent assay; inducible nitric oxide (iNOS), cyclooxygenase-2 (COX-2) and cannabinoid (CB1 and CB2 ) receptors were analysed by RT-PCR (and/or Western blot analysis); colitis was induced by dinitrobenzene sulphonic acid (DNBS). Endocannabinoid (anandamide and 2-arachidonoylglycerol), palmitoylethanolamide and oleoylethanolamide levels were measured by liquid chromatography-mass spectrometry. Colonic inflammation was assessed by evaluating the myeloperoxidase activity as well as by histology and immunohistochemistry.

Inhibitory effect of cannabichromene on nitrite levels in the cell medium of murine peritoneal macrophages incubated with lipopolysaccharide (LPS, 1 μg·mL−1) for 18 h. Cannabichromene (CBC, 0.001–1 μM) was added to the cell media 30 min before LPS challenge (i.e. 18.5 h before nitrites assay). Results are mean ± SEM of six experiments (in triplicates). #P < 0.001 versus control; *P < 0.05 and ***P < 0.001 versus LPS alone. The insert (on top of the figure) shows the effect of CBC (expressed as percentage of inhibition of the corresponding control values, with the difference between LPS and control considered as 100%) when given 30 min before LPS (CBC before LPS) or 15 h after LPS (CBC after LPS). No statistically significant difference was observed between the two concentration–response curves reported in the insert.

Inducible nitric oxide synthase (iNOS) (A, B) and cyclooxygenase-2 (COX-2) (C, D) mRNA and protein levels in cell lysates from macrophages incubated or not with lipopolysaccharide (LPS, 1 μg·mL−1) for 18 h. mRNA expression was evaluated by RT-PCR. The expression levels, normalized with respect to the reference genes, were scaled to the expression value of the control, considered as 1. The means of the quantitative-cycles (Cq) for the control were: 26.00 and 25.58 for iNOS and COX-2 respectively. The reaction background was N/A (see text) at 40 reaction cycles. Protein expression was evaluated by Western blot analysis. Cannabichromene (CBC, 1 μM) was added to the cell media 30 min before LPS challenge. #P < 0.001 versus control (n = 4–5 experiments).

Relative mRNA expression of cannabinoid CB1 receptor (A) and cannabinoid CB2 receptor (B) in cell lysates from macrophages incubated or not with lipopolysaccharide (LPS, 1 μg·mL−1) for 18 h: effect of cannabichromene (CBC, 1 μM, added to the cell media or 30 min before LPS challenge). The expression levels of mRNA, evaluated by qRT-PCR and normalized with respect to the reference genes, was scaled for all conditions to the expression value of the control, considered as 1. The means of the quantitative-cycles (Cq) for the control values were: 31.2 (CB1 receptor) and 24.48 (CB2 receptor). The reaction background was 37.30 Cq and 36.60 Cq for CB1 receptor and CB2 receptor, respectively, at 40 reaction cycles. #P < 0.001 versus control (n = 4).

Histological evaluations of inflamed and non-inflamed colons: effect of cannabichromene (CBC). No histological modification was observed in the mucosa and submucosa of control mice (A); mucosal injury induced by dinitrobenzene sulfonic acid (DNBS) administration (B); treatment with CBC reduced colon injury stimulating a regeneration of the glands (C). CBC (1 mg·kg−1) was administered (i.p.) for 2 consecutive days starting 24 h after the inflammatory insult. Histological analysis was performed 3 days after DNBS (150 mg·kg−1, intracolonically). Original magnification ×200. The figure is representative of three experiments.

Different patterns of Ki-67 immunoreactivity in the colonic mucosa of control mice (A), dinitrobenzene sulfonic acid (DNBS)-treated mice (B) and mice treated with DNBS plus cannabichromene (C). (A) Ki-67 immunopositive cells localized to the lower of the crypts. (B) Ki-67 immunoreactivity was observed on inflammatory cells. (C) Ki-67 immunopositive cells observed only in the expanded basal zone. CBC (1 mg·kg−1) was administered (i.p.) for 2 consecutive days starting 24 h after the inflammatory insult. The figure is representative of three experiments.